For many years we have been studying how polyamines are synthesized, how their biosynthesis and degradation are regulated, their physiologic functions and how they act in vivo. For this purpose we have constructed null mutants in each of the biosynthetic steps in both Escherichia coli and Saccharomyces cerevisiae, and have prepared over-expression systems for the biosynthetic enzymes. Despite many studies for years from our own and other laboratories there has been uncertainty of the specific mechanisms involved in the pleotropic effects of polyamines. Our current studies using microarray and proteomic techniques have characterized the specific mechanism for many of the polyamine effects. In our earlier microarray studies we found that within 60 minutes after polyamine addition 54 genes were up-regulated more than 2-fold and 15 genes were up-regulated more than 3-fold; 94 genes were down-regulated more than 2-fold. It was striking that most affected system involved the genes involved in the development of acid-resistance in E. coli. Not only were the two genes for glutamate decarboxylases and the gene for glutamate gamma-aminobutyrate antiporter (gadC) induced by the polyamine addition, but also the various genes involved in the regulation of this system were induced. This acid-resistance system is of importance because to colonize the gastrointestinal tract, E. coli must survive passage through the acidic environment of the stomach. We confirmed the importance of polyamines for the induction of the proteins of the acid-resistance system by direct measurement of glutamate decarboxylase protein and activity and by measuring acid-survival at pH 2.5. In the absence of added polyamines there was no induction of glutamate decarboxylase and no protection against acid stress. The protection from acid stress required polyamines, glutamic acid, and glutamate decarboxylase. The effect of deletions of the regulatory genes on this system and the effect of overproduction of three of these genes were also studied. Deletion of either rpoS (coding for the alternate Sigma factor of RNA polymerase) or gadE or rcsB resulted in complete loss of glutamate decarboxylase activity and acid resistance even in cultures containing polyamines. In conclusion, these data show that a major function of polyamines in E. coli is protection against acid stress by increasing the synthesis of glutamate decarboxylase, presumably by increasing the levels of the rpoS and gadE regulators. In our more recent studies we focused our attention on the regulatory network responsible for this polyamine induced glutamate decarboxylase activation, and also carried out preliminary studies on some other systems in which polyamines protect E. coli from other conditions of stress. For these studies we constructed a new polyamine-deficient strain containing well-defined deletion mutations in all of the genes involved in polyamine biosynthesis. Further confirmation of the regulatory roles of rpoS and gadE is shown by a comparison of genome-wide expression profiling data from a series of microarrays comparing the genes induced by polyamine addition to polyamine-free rpoS+/gadE+ cells with genes induced by polyamine addition to polyamine-free delta rpoS/gadE+ and rpoS+/delta gadE cells. The results indicate that a large percentage of the genes induced by polyamine addition (including the GDAR system) require an intact rpoS gene. Polyamine addition resulted in greater than 3-fold increased expression of 85 genes, and it also suppressed 70 genes at least three-fold. Among the pathways most affected by the addition of polyamines were the genes of the E. coli acid response pathway and genes in the acid fitness island including gadA, gadB, gadC, gadE, hdeD hdeA, slp, dctR, and yhiD; Most striking were the very large increases in gadA and gadB expression (20-fold and 26-fold), confirming our previous results. Many oof these inductions were markedly decreased if the cells contained a deletion in rpoS or gadE. To supplement the microarray data, we also carried out quantitative-real-time PCR analyses on the effect of rpoS or gadE deletions on polyamine induced expression of six of these mostly affected genes. These results confirm the microarray data that gadE and rpoS as well as polyamines are critical for the induced expression of most of the genes in the E. coli acid response system. Polyamine addition increased glutamate decarboxylase activity 50-70-fold in a stationary phase culture of rpoS+/gadE+ cells; in contrast the &#916;rpoS gadE+ and the rpoS+ &#916;gadE strains had no activity even if the culture contained polyamines. We also constructed a polyamine mutant that lacked both the rpoS and gadE genes, and repeated the above experiments in presence of prpoS, or pgadE or both. Overexpression of the RpoS protein from a plasmid had no effect in the absence of the gadE gene. In contrast overexpression of a gadE plasmid was able to partially replace an rpoS deletion. Addition of plasmids overexpressing both RpoS and GadE proteins from both plasmids plus polyamine addition resulted in a very large increase in glutamate decarboxylase activity. Hence, gadE is most directly involved in the synthesis of and activity of glutamate decarboxylase via the activation of gadA and gadB, and rpoS acts by stimulating gadE synthesis. The above conclusion on the direct involvement of gadE for the GDAR system is further supported by survival assays at pH 2.5. From the above experiments it is clear that gadE expression is directly correlated with polyamine induced expression of glutamate decarboxylase activity. Using a RT-PCR method we have used the RNA from our polyamine-free mutant strain (HT873- rpoS+/gadE+) to study the effect of polyamine addition on each of the three promoters. All the three promoters showed enhanced activity after polyamine treatment, particularly P2. We next tested the effect of an rpoS deletion (HT874-&#916;rpoS/gadE+) on the polyamine induction of gadE promoters by quantitative real time PCR. In the absence of rpoS there was no induction by polyamines of any of the three promoters of gadE. Most strikingly we found that polyamine addition markedly increased the RpoS protein level within twenty minutes after addition of polyamines to the amine-deprived culture. In the early growth phase in the absence of polyamines, there was only a trace amount of RpoS protein. However addition of polyamines for a very short time (20 min) resulted in a large increase in RpoS protein level at all optical densities. We postulate a cascade model in which the primary action of the polyamine addition to the polyamine-deficient cells is the very rapid, increase in the RpoS level with subsequent induction of gadE expression, that in turn increases the GDAR system. This observation that polyamines increase the amount of RpoS protein is a major breakthrough in understanding the physiological role of polyamines in bacteria. This finding is of importance not only for understanding the requirement of polyamines for the glutamate-dependent-acid-response system but also for the pleotropic effects of polyamines in regulating the large number of systems already known to involve RpoS. We have already confirmed this mechanism for the protective effect of polyamines against oxygen stress (H2O2) and DNA damaging agents, and for long time survival. We have also found that some effects of polyamines are not dependent on the RpoS system. As part of these studies we found a mutant (mnmE&#916;) that modifies the U34 position in tRNA, which has an absolute requirement of polyamines for growth. We have recently obtained bypass mutants to study the mechanism of polyamine action in helping growth of mnmE&#916; cells.